JP6417424B2 - Implantable paste and use thereof - Google Patents

Description

  The present invention relates to an implantable paste comprising bioactive glass for use as an implant or as a coating for an implant.

  Bioactive glasses are known as bioactive and biocompatible materials. For decades, bioactive glass has been developed as a bone filler that can be chemically combined with bone. Recent discoveries regarding the superiority of bioactive glass have deepened interest in applying this material. Bioactive glasses are commercially available, for example, under the trade names BonAlive (R), NovaBone (R), and Biogran (R). Bioactive glass is used in various forms as medical applications such as granules and plates for orthopedic and cranio-maxillofacial bone cavity filling and bone restoration It has been.

  The main advantage of using bioactive glass as a bone graft substitute is that it is possible to avoid harvesting bone graft from secondary areas. Within certain configurations, the bioactive glass promotes bone growth and exhibits bacterial growth-inhibiting properties.

  In order for a glass to be biologically active and have the aforementioned properties, it must melt and have a certain dissolution rate as well as having a certain composition. . The relationship between composition and biological activity is described in Hench.L “Theory and clinical applications” (Bioceramics). The description in Bioceramics (1994; 7: 3-14) provides those skilled in the art with sufficient means to design bioactive glasses.

One factor affecting the dissolution rate and thereby the total degradation time of the glass particles is the particle size, or the surface area (A / V) relative to the volume. In other words, the smaller the particles, the higher the A / V rate, the faster the decomposition, and the shorter the total decomposition time. For example, the commercially available glass 45S5 / Bioglass® is available in the size range of 90-710 μm and is said to degrade in the body within one year. Glass S53P4 sold under the trade name BonAlive® has a chemical composition of 53 wt% SiO ₂ , 23 wt% Na ₂ O, 20 wt% CaO and 4 wt% P ₂ O ₅ . And it is a glass that melts obviously more slowly than 45S5 glass having a composition of 45 wt% SiO ₂ , 24.5 wt% Na ₂ O, 24.5 wt% CaO and 6 wt% P ₂ O _5. .

  Deformable paste or putty type compositions have been developed to facilitate use and to extend the surgical scope of bioactive glass. In an ideal case, the putty composition should facilitate dosing, treatment of bone defects and direct management without the risk of contact contamination, spillage or overdose. In practice, physicians have used their hands for drug administration and putty formation, and have used fingers and / or spatulas or the like to fill the bone sinus. However, such a format has many practical disadvantages, such as contamination risks that are not optimal for the patient or physician.

  One synthetic putty / paste composition is known in US2008 / 0226688 and is commercially known as NovaBone® putty. The literature discloses bone-filled filler-type pastes or patties, i.e., sterilized moldable implant compositions composed of bioactive glass particles that can be applied to bone defects in aqueous media solutions. ing. Bioactive glass particles are added to the adhesive medium in a concentration range of about 68% to about 76% (wt / wt). The medium comprises a mixture of glycerol and medium molecular weight polyethylene glycol (PEG) in the range of 24% to 32% (wt / wt), wherein the ratio of glycerol to polyethylene glycol is from about 45:55 to about 65:35. .

  Putty or pasty, such as allograft bones, demineralized bone matrix, and bovine collagen / hydroxyapatite blends in addition to fully synthesized bone cavity filler putty or paste Certain semi-synthetic mixtures have been widely used in the field. However, such allograft formulations have many disadvantages that are the greatest and never completely ruled out risk of disease progression.

  In addition, prosthetic joint infections (PJIs) and other abiotic implant infections are important health care concerns, such as long-term hospitalization and additional risks associated with complex risks and long-term antimicrobial treatment. It is associated with many inconveniences for the patient. Despite the relatively low incidence of PJIs (1-2%), the associated economic impact remains significant. A wide range of microorganisms often causes a fatal series of treatments and can create a biofilm on the material of the prosthesis. Staphylococcus aureus, coagulase negative staphylococci and gram-negative rods are generally the most pathogens associated with PJIs.

  The first stage of development in PJIs is the adhesion of bacterial cells to the implant, followed by the formation of a biofilm matrix. A biofilm is a population of microorganisms surrounded by a matrix adhered by cell adhesion forces between the microorganisms and the abiotic surface. Biofilm formation is a very long-standing component of the integrated prokaryotic cycle and is an important factor for the survival of bacteria in the environment. A common feature of biofilm-related infections is intrinsic immunity to host immunity, typical antibacterials and insecticides. Bacteria surrounded in the structure of the biofilm are actually known to have an acceptable level of antibiotics 10 to 1000 times higher than the minimum inhibitory concentration of the corresponding planktonic organism.

  A further common problem associated with bacterial infection is the ability of bacteria to be resistant to antibiotics. It is not a recent phenomenon, but it is a serious health problem today. Over the decades, to varying degrees, highly infectious bacteria have developed resistance to each new antibiotic, and according to the US and World Health Organization's Centers for Disease Control and Prevention, antimicrobial resistance is worldwide. It has become a healthy threat.

  Indeed, infection from resistant bacteria is now too frequent, and some pathogens have been able to resist even multiple types or classes of antibiotics. Today, antibiotic resistance is seen in most hospitals, and it is estimated that 70% of all bacteria will be resistant to antibiotics by 2020. Methicillin resistant S. aureus (MRSA), Methicillin resistant Staphylococcus epidermidis (MRSE), Pseudomonas aeruginosa, and Acinetobacter (Pseudomonas aeruginosa) isolated from patients infected with chronic osteomyelitis In vitro bacterial growth inhibitory activity of BAG S53P4 against Acinetobacter baumannii has been shown (Drago L, Romano D, De Vecchi E, et al. Bioactive glass BAG-S53P4 for the adjunctive treatment of chronic osteomyelitis of the long bones. An in vitro and prospective clinical study. BMC Infect. Dis. 10, 13: 584 (2013)). However, although it is known that the small particles of this bioactive glass have an antibacterial effect. There is no known practical interaction between them and other components.

  The document "Antibacterial effect of bioactive glasses on clinically important anaerobic bacteria in vitro" (J. Mat. Sc. In Medicine, Vol. 19, No. 2, 2007/7/10) has the same size or less than 45 μm. Discloses that the powder of bioactive glass S53P4 has a good antibacterial effect. Applicant's document EP2322134 discloses an implant paste having different types of polyethylene glycol and bioactive glass spheres. However, as shown below, these pastes are not antimicrobial.

US Patent Application Publication No. 2008/0226688 European Patent No. 2322134

(Definition)
The terms used in this application are those agreed at the consensus conference on biological materials in 1987 and 1992, if not defined.
Reference: Williams, DF (ed.): Definitions in biomaterials: Proceedings of a consensus conference of the European Society for Biomaterials, Chester, England. March 3-5, 1986. Elsevier, Amsterdam 1987, and Williams DF, Black J, Doherty PJ.Second consensus conference on definitions in biomaterials.
In: Doherty PJ, Williams RL, Williams DF, Lee AJ (eds) .Biomaterial-Tissue Interfaces.Amsterdam: Elsevier, 1992.

  In this application, a bioactive material means a material that has been designed to elicit or modulate bioactivity. Bioactive materials are surface active materials that are often capable of chemically binding to mammalian tissue.

  The term resorbable in the text means that the material is disintegrated, i.e., degraded in long-term transplantation when placed in the mammalian body and in contact with the physiological environment. In particular, the term resorbable glass means a silica-rich glass that does not form a hydroxyl-carbonate apatite layer on its surface when not in contact with a physiological environment. Absorbent glass disappears from the body by reabsorption and does not significantly activate cells or cell growth during the degradation process.

  By biomaterial is meant a material intended as an interface to a biological system for evaluation, processing, expansion, or replacement of any tissue, organ or function of the body. Biocompatibility refers to the performance of materials used in medical devices that function safely and well by inducing appropriate host reactions at specific locations. Resorption means the degradation of biological material by simple decay. By composite is meant a material such as glass that has at least two different components, such as organic polymers and ceramic materials.

  It is an object of the present invention to provide a mixture that can be easily and safely processed and is useful as a bone cavity filler with the desired bone filling properties as well as antibacterial properties. Another object of the present invention is to provide a composition useful for preventing biofilm formation on the surface of an implant. The object of the invention is therefore also to provide a material for use in implant surgery to prevent local infection. A further object of the present invention is also to provide a material that will not gain resistance.

The present invention relates to an implant paste having bioactive glass granules having a size distribution of 0.5 to 45 μm and a size distribution of 100 to 4000 μm.
The composition of the bioactive glass _SiO 2 45 to 55 _wt%, Na 2 O 20-25% by weight, CaO 18 to 25 wt% and _P 2 _O 5 3 to 6 wt%. The paste further comprises 200-700 g / mol, low molecular weight polyethylene glycol, 700-2500 g / mol medium molecular weight polyethylene glycol, 2500-8000 g / mo high molecular weight polyethylene glycol, and glycerol. However, the molecular weight of the low molecular weight polyethylene glycol and the molecular weight of the medium molecular weight polyethylene glycol are different from each other by at least 80 g / mol, and the molecular weights of the medium molecular weight polyethylene glycol and the high molecular weight polyethylene glycol are different from each other by at least 300 g / mol.

The invention also relates to the use of a paste used in the manufacture of an implant of the invention for use in bone formation.
The invention further relates to the use of a paste for the coating of the implant according to the invention.
The invention further relates to an implant covered with the paste of the invention.

The present invention
(A) a bioactive glass powder having a size distribution of 0.5 to 45 μm;
(B) bioactive glass granules having a size distribution between 100 and 4000 μm;
(C) a low molecular weight polyethylene glycol having a molecular weight range of 200 to 700 g / mol;
(D) a medium molecular weight polyethylene glycol having a molecular weight range of 700-2500 g / mol;
(E) high molecular weight polyethylene glycol having a molecular weight range of 2500 to 8000 g / mol, and (f) glycerol.
Wherein the bioactive composition _SiO 2 45 to 55 _wt% of the glass, Na 2 O 20-25 wt%, CaO 18 to 25 wt%, and a _P 2 _O 5 3 to 6 wt%, about implantable paste. However, the molecular weight of the low molecular weight polyethylene glycol and the molecular weight of the medium molecular weight polyethylene glycol are different from each other by at least 80 g / mol, and the molecular weights of the medium molecular weight polyethylene glycol and the high molecular weight polyethylene glycol are different from each other by at least 300 g / mol.

  The present invention thus provides an osteoconductive, bioactive, fungal resistant and deformable composition. The paste has a sticky organic medium solution or bioactive glass granules in a matrix. The paste is therefore composed of calcium-phosphorous-sodium-silicate particles mixed with a synthetic binder composed of polyethylene glycol which acts as a temporary binder for the particles. Yes. The particles and binder are typically provided as a premixed cohesive material. In transplantation, the binder is absorbed to allow tissue penetration between the particles and to allow the normal healing process of bone bound to the particles (resorption of bioactive glass and bone regeneration). If the binder is absorbed immediately after implantation, only bioactive glass particles remain. All these components are known and widely used and are approved in the medical, pharmaceutical and cosmetic fields as well as in the food and beverage fields.

  At least some of the above objects will be met by the invention and its various embodiments, if not at least some of the above objects. In fact, when the fine bioactive particle powder is combined with a polyethylene glycol mixture, the resulting material produces surprising antibacterial properties while simultaneously promoting bone ingrowth.

  European patent EP2322134 discloses a similar composition comprising a sphere of bioactive glass and a mixture of different polyethylene glycols (low, medium and high molecular weight PEG). However, it has been seen that compositions in which the bioactive glass is BonAlive® bioactive glass have no significant antimicrobial effect. On the other hand, BonAlive® bioactive glass has been shown to have an antibacterial effect regardless of its particle size, even against antibiotic resistant bacteria. However, fine bioactive particles (average diameter less than 45 μm) alone cannot be incorporated into actual mammalian tissue because of their high reaction rate and potentially high risk. It will also not promote bone growth because of its high response rate.

  Compared to bioactive glass powders of similar particle size, the present material comprising polyethylene glycol has the additional advantage that it can be applied through a tube and can also be used for small incision surgery. The material is tacky and fills the defects in a controlled and gradual manner as opposed to a free flowing dry powder. Therefore, it is possible to control the stable supply of paste to the defect.

  It is also possible to combine bioactive glass with a larger granule shape from a small particle size powder, which will affect both the bone ingrowth properties and the material flow properties. The choice of polyethylene glycol will be applied to meet the requirements for flow properties.

  In this specification, the abbreviation wt% means weight percent and is typically expressed as weight percent of total weight. Molecular weight means the mean molecular weight, here the number average molecular weight, expressed as g / mol. The size distribution of the bioactive glass particles is determined by sieving.

  By granule is meant any regular or irregularly shaped particles other than spherical. Bioactive glass powders are also composed of granules.

According to a preferred embodiment, the paste is
(G) a therapeutically active agent,
including.

  According to one embodiment, the bioactive glass granules have a size distribution of 500-800 μm. According to another embodiment, the bioactive glass granules have a size distribution of 100-350 μm. According to yet another embodiment, the bioactive glass granules have a size distribution of 315-500 μm. According to another embodiment, the bioactive glass granules have a size distribution between 1000 and 2000 μm. The composition may comprise bioactive glass, for example in the form of powders and granules having a size distribution of 315 to 500 μm. Such a composition is useful, for example, for oral surgery. Another possible combination is a powder and granules having a size distribution of 500-800 μm. This composition is beneficial for craniomaxillofacial and hand surgery. A further possible combination is a granular powder with a size distribution of 1000 to 2000 μm. This composition is useful for orthopedic surgery, trauma and spinal surgery.

  The composition may thus comprise a bioactive glass in powder form, for example in granules having a size distribution of 1 to 44 μm. The size distribution can be, for example, from 1, 5, 10, 15, 20, 25, 30, 35 or 40 μm to 5, 10, 15, 20, 25, 30, 35, 40 or 45 μm. Further, the composition may have a size distribution of, for example, 100-350 μm, alternatively 100-200 μm, alternatively 150-250 μm, alternatively 200-300 μm, alternatively 250-350 μm. Larger granule size distributions may be, for example, 315-500 μm, alternatively 350-500 μm, alternatively 400-500 μm. Further, the size distribution of the larger granules may be, for example, 500-800 μm, alternatively 500-700 μm, alternatively 550-800 μm, alternatively 600-800 μm, alternatively 650-750 μm. The size distribution may also be in some embodiments, for example, 1000-2000 μm, alternatively 1000-1500 μm, alternatively 1300-1800 μm, alternatively 1500-2000 μm, alternatively 2000-3150 μm. The size distribution of the glass granules is determined by sieving.

  The size distribution of the glass granules can also be, for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, From 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2900, 3000 or 3150 μm, 150 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2900, 3000, 3100, 3150, 3200, 3300, 3400, 3450, 3550, 3600, 3700, It may be up to 3800, 3900 or 4000 μm.

Polyethylene glycol (PEG) is known as a polyethylene oxide containing a repeating unit (—CH ₂ CH ₂ O—), and is obtained by sequentially adding ethylene oxide to a composition containing a reactive hydrogen atom. . Polyethylene glycol is obtained by adding ethylene oxide to ethylene glycol so as to constitute a bifunctional polyethylene structure HO (CH ₂ CH ₂ O) _n H, where n is an integer matched to the molecular weight of polyethylene glycol Is

    The polyethylene glycol used in the present invention is a general linear polyethylene glycol having a molecular weight of 100 to 8000 g / mol. Branched polyethylene glycol can also be used to reduce or adjust the viscosity of the paste. Polyethylene glycol is typically indicated with a number as PEG, the number meaning the average molecular weight in g / mol. Accordingly, PEG400 means polyethylene glycol having a molecular weight of 400 g / mol, and PEG2000 means polyethylene glycol having a molecular weight of 2000 g / mol.

  Polyethylene glycol (PEGs) is used to form pasty materials by binding and wetting bioactive glass particles. In order to achieve the proper viscosity of the paste, at least two PEGs are mixed. In selecting an appropriate molecular weight for PEGs, it should be noted that low molecular weight PEG (<600 g / mol) is liquid at room temperature, while higher molecular weight PEGs are waxy or solid.

    In order to maintain the paste in a paste at its operating temperature (room and body temperature), at least three PEGs are mixed and typically kept at a high temperature. When higher molecular weight PEGs are crystalline materials, they increase the paste's use temperature, as well as increase the viscosity of the paste and prevent the precipitation of bioactive glass particles in a room temperature storage. Can do. In order to lower the lower limit level of use temperature, that is, in order to widen the use temperature range, high molecular weight PEGs tend to crystallize at low temperature, so low molecular weight PEGs avoid solidification of paste, that is, paste cure at low temperature. Is beneficial for.

  If a single molecular weight waxy or solid PEG is used, the operating temperature range may be too narrow for practical use. PEG 600 (ie, polyethylene glycol with a molecular weight of 600 g / mol) exhibits a melting range of about 17-22 ° C., so it will be liquid at room temperature and pasty at lower ambient temperatures. On the other hand, PEGs having an average molecular weight of 800 to 2000 are pasty materials having a low melting range. When the average molecular weight is above 3000, the polyethylene glycol is typically a solid.

  Glycerol, ie propane-1,2,3-triol, is usually called glycerin or glycerine. Widely used in formulations are colorless, odorless and sticky liquids. Glycerol may be added to improve smoothness and to provide additional lubricity with increasing temperature and viscosity due to physical interaction between PEGs and glycerol. PEGs and glycerol can be used together.

  PEG 400 is miscible in all properties with glycerol, but the degradation power and solubility of PEG in glycerol decreases with increasing molar concentration. However, both of these properties can be improved by gentle heating, and a material that dissolves in PEG 400 at room temperature is approximately as soluble in dissolved PEG 4000 (ie, at 60-70 ° C.).

  According to one embodiment of the present invention, the total amount of bioactive glass is 50-80 wt% of the total weight of the paste. The total amount (a) of the bioactive glass having a size distribution of 0.5 to 45 μm is 10 to 30 wt% of the bioactive glass, and the bioactive glass granules having a size distribution of 100 to 4000 μm. The total amount (b) is 90 to 70 wt% of the total weight of the bioactive glass in the paste.

  Preferably, the total amount of polyethylene glycol is 20-50 wt% of the total weight of the paste. For example, the total amount (c) of low molecular weight PEG is 2 to 15 wt%, and the total amount (d) of medium molecular weight PEG is 8 to 48 wt% of the total weight of the paste. The total amount (e) of the high molecular weight PEG is 1 to 10 wt% of the total weight of the paste.

In embodiments where glycerol is used, the total amount is up to 10 wt% of the total weight of the paste. Some suitable pastes are formulated as follows.
PEGs (c + d + e) 23-45 wt
Glycerol (f) 0-10 wt%
Bioactive glass (a + b) 55-67 wt%

Some preferred pastes have the following formulation ranges.
Low molecular weight PEG (c) 4-10 wt%
Medium molecular weight PEG (d) 13-18 wt%
High molecular weight PEG (e) 1-8wt%
Glycerol (f) 8-10 wt%
Small bioactive glass granules (a) 8-12 wt%
Larger bioactive glass granules (b) 48-52 wt%

  According to an embodiment of the invention, the total amount (g) of therapeutically active agent (therapeutically active agent) is at most 30 wt% of the total weight of the paste. The therapeutic active agents are composed of growth factors, proteins, peptides, antibiotics, mucopolysaccharides such as hyaluronic acid, non-human stem cells (ie, other than human stem cells), peroxides and mixtures thereof. Can be selected from these groups. The active agent can then be used to promote bone growth and acquire additional antibacterial properties such as antibacterial effects. This material, however, has been found to be effective in the absence of antibiotics.

According to an embodiment of the present invention, the composition of the bioactive glass _SiO 2 45-54 _wt%, Na 2 O 22-25 wt%, CaO 19 to 25 wt% and _P 2 _O 5 3.5 to 6 wt %. According to another embodiment of the present invention, the composition of the bioactive glass product is 53 wt% SiO ₂ , 23 wt% Na ₂ O, 20 wt% CaO and 4 wt% P ₂ O ₅ . Such a bioactive glass is also known as S53P4 and is also commercially available under the trade name BonAlive®. This embodiment provides a binder composition that dissolves rapidly in vivo to allow the normal bone treatment process to combine with S53P4 granules and powder (bioactive glass and bone resorption resorption). Due to the slow degradation rate and particle size of the S53P4 bioactive glass composition, a long-term bone growth effect can be naturally achieved. According to yet another embodiment, the bioactive glass composition _SiO 2 _45% by weight, Na 2 O 24.5% by weight, CaO 24.5 wt% and _P 2 _{O 5} 6 wt%. This bioactive glass is also known as 45S5 and is commercially available as NovaBone®.

  Pastes composed of components at the ends of the range do not necessarily give optimum flowability and product characteristics. For example, a combination of a high concentration of high molecular weight PEG in the absence of sufficient low molecular weight PEG and / or glycerol gives the product a high viscosity but is not suitable for implantation at room or body temperature. However, one of ordinary skill in the art can find the ideal proportion of ingredients through some simple experimentation for each desired combination of properties. Some examples of suitable combinations are also provided in the example part described below.

  The present invention also provides a method for producing a deformable bone void filling paste having antibacterial properties, which melts and mixes controlled controlled raw materials as well as cooling, packaging and final product preparation. Including.

  Pastes are typically made by mixing and / or melting the materials in a batch mixer in a protective mixer or vacuum or atmospheric atmosphere at 25 to 90 ° C. for 5 to 60 minutes. The mixture is then cooled to 25-45 ° C. and transferred to an applicator and / or stored for further use. Alternatively, mixing, melting, and / or transfer may be performed by, for example, an open or closed batch mixer, a continuous stirring tank reactor or mixer, an extruder. Any type of mixing device known in the art, such as an injection molding machine, a tube reactor, or other standard melt processor or melt mixing equipment Can also be implemented.

  The present invention also provides the use of this paste in the manufacture of implants for use in the formation of bone in bone defects such as bone cavity filler paste. In fact, the paste has been determined to have the ability to prevent and treat biofilm formation on the surface of the implant. It has an antibacterial effect against the bacteria that are the most problematic of prosthetic device infections and can therefore be used to treat biofilm-related prosthetic device infections. Furthermore, the pastes herein can be used to treat wounds by being applied over and covering the open wound.

  The present invention also provides an implant coated with the paste. The implant can be employed, for example, as a hip implant, knee implant or any other artificial joint, or any other implant employed in mammals. The implant is coated with this paste before being implanted into the human or animal body.

  In addition, the present invention provides an antibacterial bone growth promoting composition comprising an agent having a therapeutic activity as described above (active agent). The active agent may be any pharmaceutically active agent used for humans or animals.

  Different embodiments of the invention are more fully described in the following example part.

(General manufacturing method for putty)
Glycerol and PEG400 were added to a reaction vessel (60 ° C.) with a mixing speed of 100 prm (reaction / min), followed by PEG1500 and PEG3000. PEGs were provided by Clariant and glycerol was provided by Uniqema or Sigma-Aldrich. Bioactive glass granules were added to the melted mixture and mixed until the mixture was uniform. The resulting putty was cooled to room temperature (RT) with mixing. The container was then removed and stored in a dryer for further use and testing.

(Antimicrobial activity of bioactive glass)
The bactericidal performance of the bioactive glass alone and when present in the matrix described herein as a reference example was tested. The bacteria used in the test are shown in Table 1. MetR means methicillin resistance. The various glasses tested are shown in Table 2. Glass S53P4 and BonAlive (registered trademark) glass have the same composition as described above.

BonAlive® Putty Composition

Glycerol 16g
PEG400 12.8g
PEG 1500 25.6g
PEG 3000 9.6 g
500-800 μm S53P4 granular 76.8 g
90-425 μm S53P4 spherical 9.2 g

  PEG means polyethylene glycol, and the number at the end indicates the average molecular weight g / mol. A similar mixture of glycerin, PEG400, PEG1500 and PEG3000 (ie a pasty binder) was used for putty 1 to putty 6, which is a reference sample for the BonAlive® putty.

  Putty compositions from putty 1 to putty 6 were tested with Staphylococcus epidermidis and Pseudomonas aeruginosa, and all other compositions were tested with all strains listed in Table 1.

In vitro test (Comparative Examples 1-4, 11-14)
Bacteria were cultured with different products (5 ml sterile) in sterilized tryptone soy medium (TSB consisting of casein enzyme digest, soybean meal enzyme digest, sodium chloride, dipotassium phosphate and glucose). Test tube, Becton Dickinson). The concentration of the product used in the test is shown in Table 3.
Granules, comparative materials and controls were weighed (Mettler AE 50) and mixed appropriately with 2 ml TSB. Three replicates were weighed except for 6 replicates of each putty product. Three replicates of the putty product were incubated at room temperature for 2 hours, after which the TSB was replaced with a new 2 ml batch of TSB along with the dissolved polymer. Finally, bacterial inoculum (absorbance meter, known amount measured by hermo GeneSys 20) was added to the mixture. Bacterial cultures without added product and pure TSB were used as controls. Although S53P4 powder serves as a positive control, it is known from recent studies that powder at a concentration of 100 mg / ml is effective for the bacterial inhibitory effect. Iittala glass and tricalcium phosphate (TCP) were added to the comparative materials. The inert Iittala non-bioactive glass was ground into fine granules (exact particle size unknown) prior to testing.

  Viability of bacterial suspensions cultured with different products was evaluated using a commercially available solid plasma agar plate (Trypticase Soy Agar II with 5% Sheep Blood, Becton Dickinson). A 10 μl sample taken directly from the suspension at 24 hours of continuous culture was plated (Vuorenoja K, Jalava J, Lindholm L. et al. (2011) Detection of Streptococcus pneumoniae carriage by the Binax Eur J Clin Microbial Infect. EPub PMID: 21800217)) NOW test with nasal and nasopharyngeal swabs in young children. In addition, 1: 1000 sample dilutions and bacterial controls were plated to confirm quantitative, single colonies.

  Bacterial growth was assessed by comparison with control samples after incubation on agar plates (16 hours at 37 ° C.). The absence of growth on the plate was a performance indicator given to the product to prevent bacterial colonization. In vitro culturing was carried out for 7 days, except for P. aeruginosa, where the formation of viscous material hinders accurate collection of 10 μl samples, which may not exceed 5 days.

  The pH of the sample was measured from the test tube using pH paper (pH 7.5-14 range, Merck Alkalit 81.09532 and pH 6.4-8.0 range Nacherey-Nagel, REF 90210). The pH value was measured by immersing the paper in the culture medium and comparing it with the scale colored paper provided by the manufacturer. The pH measurement was performed after 8 days of culture in S. aureus and S. epidermidis, 7 days in MRSA, and 5 days in P. aeruginosa.

In vitro test (Reference Examples 5 to 10)

One strain of Staphylococcus epidermidis and one strain of Pseudomonas were used. Each product (final concentration 400 mg / ml) was conditioned through incubation in growth medium at 37 ° C. for 48 hours. The pH value was measured at regular intervals. A pH value equal to or higher than 10 was assumed to be the optimal adjustment condition.

  Antibacterial activity was assessed using killing curves. A portion of the bacterial suspension was inoculated into a test tube containing the conditioned product. Growth adjustment was carried out by bacterial inoculation of the growth medium only. Tubes were incubated at 37 ° C. in an aerobic atmosphere. Microbial counts were performed by culturing appropriate dilutions of bacterial suspensions cultured at 37 ° C. for 24 hours for 0, 24, 48 and 72 hours.

Results of Comparative Materials (Comparative Examples) (Reference Examples 1-4, 11-14)

The negative control consistently showed too many results to count after culture. This was seen in all lines and at all time points, indicating that the bacteria were viable throughout the test period. Pure TSB without inoculum was used as a control to indicate that all work was performed under aseptic conditions. There was no bacterial growth in pure TSB at any time.

  All BonAlive® granules tested alone affected bacterial growth. The time and level required to obtain the effect varied depending on the granule size. The effect was also different depending on the bacterial species. Powders sized <45 μm were resistant to the growth of all tested bacteria. The BonAlive® putty product did not affect the growth of any of the pathogens tested. S.epidermidis, S.aureus MetR and P.aeruginosa results showed a slight effect during the study period, but BonAlive® putty did not affect S.aureus ATCC 29213 . There was day-to-day variation during the test period. The pure polymer (putty binder) had some effect on S. aureus MetR and P. aeruginosa at the end of the test period, but growth varied from day to day. TCP had no effect on gram negative P. aeruginosa on any day, nor did it affect S. aureus ATCC 29213 at the end of the study period. A slight effect was observed on two other Gram-negative bacteria.

  It can be said that the BonAlive® putty product failed to block the colonization of four clinically important pathogens during the study period. The daily variability and indeterminate results of putty and polymer can be explained at least in part by in vitro testing. In addition, the polymer and the chemical characteristics of the polymer may have any impact on pipetting quality.

The results of bacterial growth are shown in Tables 4-7. The numbers shown are the difference between the bacterial control and the cultured bacteria (log ₁₀ ) in the sample (0 = no difference to the bacterial control, 1 = log ₁₀ difference to the bacterial control, 5 = 4 log difference or more ). 0 means no inhibition on bacterial growth.

  Table 8 shows the pH values at the end of the culture period (8 days for S. epidermidis and S. aureus, 7 days for S. aureus MetR, 5 days for P. aeruginosa).

Reference examples 5-10
The results of Comparative Examples 5 to 10 are shown in Table 9. The putty composition showed a slight pH change (pH 8) but had no antibacterial effect on S. aureus and P. aeruginosa.

Antibacterial activity of the paste of the present invention

The paste with the formulation shown in Table 10 (Composition 1) was tested for antimicrobial activity. The comparative sample contained 45 μm size fraction bioactive glass powder (Composition 2) and 500-800 μm size fraction bioactive glass granules (Composition 3) that were gamma sterilized as described above. It was.

  Three samples of similarly sized inert glass (clear, R1350, Iittala, Finland) were used as negative controls. Table 9 shows PEG and glycerol, <45 μm granules (Comparative Example 1), 45 μm granules (Comparative Example 2), and 500-800 μm granules (Comparative Example 3). All samples were prepared in tryptone soy medium (TSB; Biomerieux, Marcyl 'Etoile, France) to a final concentration of 400 mg / ml (corresponding to 5% of the clinical dilution standard solution). 4.8 ml was placed in a sterile 6-well polystyrene microplate (Jet Biofil; Guangzhou, China). Bioactive glass samples were incubated at 37 ° C. for composition 4 hours, composition 2 for 7 hours, and composition 3 for 24 hours. The pH value was measured with a pH meter under conditions where ion release and pH change were presented at regular intervals. A pH value of 11 or higher is considered to be an appropriate presented condition. Once the optimal conditions were reached, the contents of each well were ready for use.

  One strain of S. aureus resistant to methicillin and one strain of P. aeruginosa were used. It was isolated at the Microbiology Laboratory of IRCCS Galeazzi Orthopaedic Institute and from the patient's knee artificial bone associated with the Center for Reconstructive Surgery of Osteoarticular Infections (CRIO) . These strains were selected in vitro for their ability to form a biofilm on artificial bone material.

Titanium discs (Adler Ortho, Cormano (Milan), Italy; BATCH J04051) having a diameter of 25 mm and a thickness of 5 mm were used as adherends for biofilm formation and growth. Overnight cultured S. aureus and P. aeruginosa were resuspended in TSB and a portion of each diluted standard solution (200 μl) at a final density of 1.0 × 10 ⁸ CFU / ml, and 4.8 ml of titanium disk. Were inoculated into a sterile 6-well polystyrene microplate containing new TSB. After aerobic culture at 37 ° C. for 24 hours, the growth medium containing exhausted non-viscous bacteria was removed and replaced with fresh 5 ml medium. Plates are incubated for an additional 48 hours to obtain mature biofilm, and any remaining non-adherent bacteria are removed by three washes with sterile saline. did.

  After conditioning time, the titanium discs covered with bacterial biofilms were placed in a new sterilized 6-well polystyrene microplate with conditioned biological glass or negative control (inert glass). . The amount of biofilm on each titanium disc was measured after 24, 48 and 72 hours of incubation.

Crystal violet assay

The crystal violet assay was used as a primary test to evaluate the optimal formulation and the optimal incubation time for biofilms against S. aureus and P. aeruginosa. To evaluate the effect of tested glass on biofilm tissue, Christensen et al. (Christensen GD, Simpson WA, Younger JJ, et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for The amount of biomass present on each disc after treatment as described in the adherence of staphylococci to medical devices. J. Clin Microbiol 22 (6), 996-1006 (1985)) was measured. At the end of the incubation time, the biofilm grown on the titanium disc was dried with air and the disc was immersed in a 5% crystal violet solution for 15 minutes and colored. In addition, after several washes, the discs were again dried with air and the crystal violet bound to the biofilm was extracted into 3 ml of 96% ethanol. Three aliquots (100 μl) of each ethanol-dye solution were placed in a 96 multiwell plate and the absorbance at 958 nm was read with a microplate reader (Multiskan FC, Thermo Scientific; Milan Italy).

Statistical analysis

The amount of biofilm as measured by the crystal violet assay was expressed as mean standard deviation (mean ± SD). Statistical analysis was performed appropriately using the two-factor ANOVA method followed by the Bonferroni t-test and Student's t-test. The important range of p-value was set to 0.05 or less.

result

The biofilm resistance of S53P4 is shown in Tables 11 and 12 as absorbance at 595 nm of two bacteria. The amount of biofilm present on all titanium discs was significantly different (P <0.05) from the S53P4 treated disc and the inactive glass treated disc (control). Although the exposure time to the bioactive glass did not appear to affect the amount of biofilm, a decrease in biofilm was observed after 72 hours of treatment compared to observation after 24 and 48 hours. The biofilm resistance of S53P4 was not significantly different between the three formulations tested.

The results show that when mixed with glycols and PEGs, the antimicrobial activity of the bioactive glass itself is not relevant and the antibiotic effect unless a powder with a size distribution of 0.5-45 μm is used. Did not have. Therefore, since only this bioactive glass (eg powder) has a surprising effect, the combination taught by EP2322134 and the fact that the bioactive glass has antimicrobial activity is not obvious to those skilled in the art.

Claims (14)

(A) a bioactive glass powder having a size distribution of 0.5 to 45 μm and containing 10 to 30% by weight of the total weight of the bioactive glass;
(B) a bioactive glass granule having a size distribution between 100 and 4000 μm and comprising 90-70% by weight of the total weight of the bioactive glass in the paste ;
(C) a low molecular weight polyethylene glycol having a molecular weight range of 200 to 700 g / mol;
(D) a medium molecular weight polyethylene glycol having a molecular weight range of 700-2500 g / mol;
(E) high molecular weight polyethylene glycol having a molecular weight range of 2500 to 8000 g / mol, and (f) glycerol.
Wherein the bioactive composition _SiO 2 45 to 55 _wt% of the glass, Na 2 O 20-25 wt%, a CaO 18 to 25 wt%, and _P 2 _O 5 3 to 6 wt%,
However, the implantable paste wherein the molecular weight of the low molecular weight polyethylene glycol and the molecular weight of the medium molecular weight polyethylene glycol are different from each other by at least 80 g / mol, and the molecular weights of the medium molecular weight polyethylene glycol and the high molecular weight polyethylene glycol are different from each other by at least 300 g / mol. further,
(G) a therapeutically active agent,
The paste according to claim 1. The bioactive glass granules are
(B) The paste according to claim 1 or 2, which has a size distribution of 125 to 315 µm. The bioactive glass granules are
(B) The paste according to any one of claims 1 to 3, which has a size distribution of 315 to 500 µm. The bioactive glass granules are
(B) The paste according to any one of claims 1 to 4, which has a size distribution of 500 to 800 µm. The bioactive glass granules are
(B) The paste according to any one of claims 1 to 5, which has a size distribution of 1000 to 2000 µm. The paste according to any one of claims 1 to 6 , wherein the total weight of the bioactive glass is 50 to 80% by weight of the total weight of the paste. 2-15% by weight of low molecular weight polyethylene glycol (c) having a molecular weight range of 200-700 g / mol,
The amount of medium molecular weight polyethylene glycol (d) having a molecular weight range of 700 to 2500 g / mol is 8 to 48% by weight of the total weight of the paste;
The paste according to any one of claims 1 to 7 , wherein the amount of the high molecular weight polyethylene glycol (e) is 1 to 10% by weight of the total weight of the paste. The total amount of polyethylene glycol is 20-50% by weight of the total weight of the paste,
Glycero - le amount of (f) is up to 10% by weight of the total weight of the paste, the paste according to any one of claims 1 to 8. 10. Paste according to any one of claims 2 to 9 , wherein the therapeutically active agent (g) is up to 30% by weight of the total weight of the paste. The composition of the bioactive glass _SiO 2 45 to 55 _wt%, Na 2 O 22-25 wt%, CaO 19 to 25 wt%, and 1 to claim a _P 2 _O 5 3.5 to 6 wt% The paste according to any one of 10 . The composition of the bioactive glass is SiO ₂ 53 wt%, Na ₂ O 23 wt%, CaO 20 wt% and P ₂ O ₅ 4 wt%, or SiO ₂ 45 wt%, Na ₂ O 24. .5 wt%, CaO 24.5 wt%, and _P 2 _{O 5} 6 wt% in the paste of claim 11. Use of the paste according to any one of claims 1 to 12 for use in the coating of bone formation or implants. Implants coated with the paste according to any one of claims 1 to 12.